The principle goal of this project is to identify the cellular states and signals that control cell types and synapses in the central nervous system. The identification of stem cells in the central nervous system has led to the realization that large numbers of stem cells are present during brain development and that they share many differentiation mechanisms with stem cells present in the adult brain. In the past year we have reported on the analysis of anew protein that is specifically expressed in stem cells that may be involved in the mechanisms that regulate their self-renewal. We identified a novel protein, nucleostemin, found in the nucleoli of CNS stem cells, embryonic stem cells, several cancer cell lines, and preferentially expressed by other stem cell-enriched populations. It contains a N-terminal basic domain and two GTP-binding motifs. When stem cells differentiate, nucleostemin expression decreases rapidly prior to cell-cycle exit both in vitro and in vivo. Depletion or overexpression of nucleostemin reduces cell proliferation in CNS stem cells and transformed cells. Mutation analysis indicates that excessive nucleostemin, particularly mutants that lack the GTP-regulatory domain, prevents cells from entering mitosis and causes apoptosis in a p53-dependent manner. A N-terminal basic domain specifies nucleolar localization, the p53 interaction and is required for the cell death caused by overexpression. There is great interest in a molecular signature for stem cells. Gene array experiments demonstrate that previously undescribed genes are expressed in stem cells. However these studies simply describe patterns of gene expression using array techniques and the different approaches identify different sets of genes. In this manuscript we go further and clearly demonstrate the distribution of nucleostemin using Northern blots and anti-NS antibodies. We use the power of the CNS stem cell system to define the timing of nucleostemin down regulation at high resolution and we use a series of experiments to explore nucleostemin functions. This work defines the expression and function a new gene that is highly expressed in stem cells. Of course there are further experiments that will extend our understanding of nucleostemin but we believe that this study is one of the first clear examples of the use of molecular methods to study mechanisms of self-renewal.